Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells

  • Rory P. Cooney
  • Natalie J. Garton
  • Michael R. Barer
Protocol
Part of the Methods in Molecular Medicine book series (MIMM, volume 54)

Abstract

Although light microscopy fell out of favor as a research tool in prokaryotic biology in the 1980s, advances in the reagents available for cell labeling (staining) and in the user-friendliness of microscopes were underpinning a revolution in eukaryotic cell biology. The development of epifluorescence hardware, particularly confocal microscopy and low-light imaging systems, and computational deblurring and video enhancement methodologies, substantially extended the range of potential applications. These developments now enable us to detect weaker signals at higher levels of resolution than was previously possible. Finally the personal computer and related software developments have brought image analysis within affordable range for many laboratories and facilitate quantitation of cellular properties on an objective basis. We have sought to apply these advances across a range of prokaryotic applications and here we describe the methods we have applied to live Mycobacterium tuberculosis cells. Although we have principally been concerned with two applications, the determination of viability at the cellular level (see Note 1) and the nature and distribution of lipid domains, more general aspects of light microscopic cytological analyses are discussed below.

Keywords

Acetone DMSO Petroleum Chloroform Rubber 

References

  1. 1.
    Whiteley A. S., Grewal R., Hunt A., and Barer M. B. (1998) Determining biochemical and physiological phenotypes of bacteria by cytological assay, in Digital Image Analysis of Microbes (Wilkinson M. H. F. and Schut F., eds.), John Wiley and Sons, Chichester, pp. 281–307.Google Scholar
  2. 2.
    Christensen H., Garton N. J., Horobin R. W., Minnikin D. E., and Barer M. R. (1999) Lipid domains of mycobacteria studied with fluorescent molecular probes. Mol. Microbiol. 36, 1561–1572.CrossRefGoogle Scholar
  3. 3.
    Barer M.R. (1991) New possibilities for bacterial cytochemistry: light microscopical demonstration of beta-galactosidase in unfixed immobilized bacteria. Histochem. J. 23, 529–533.CrossRefPubMedGoogle Scholar
  4. 4.
    Walker D. R., Nwoguh C. E., and Barer M. R. (1994) Amicrochamber system for the rapid cytochemical demonstration of beta-galactosidase and other properties in pathogenic microbes. Lett. Appl. Microbiol. 18, 102–104.CrossRefPubMedGoogle Scholar
  5. 5.
    Paddock S. W. (1999) Confocal microscopy methods and protocols, in Methods in Molecular Biology, vol. 122. Humana, Totawa, NJ, 446 pp.Google Scholar
  6. 6.
    Matsuyama T. (1984) Staining of living bacteria with rhodamine-123. FEMS Microbiol. Lett. 21, 153–157.CrossRefGoogle Scholar
  7. 7.
    Resnick M., Schuldiner S., and Bercovier H. (1985) Bacterial-membrane potential analyzed by spectrofluorocytometry. Curr. Microbiol. 12, 183–185.CrossRefGoogle Scholar
  8. 8.
    Bercovier H., Resnick M., Kornitzer D., and Levy L., (1987) Rapid method for testing drug-susceptibility of mycobacteria spp and gram-positive bacteria using rhodamine-123 and fluorescein diacetate. J. Microbiol. Met. 7, 139–142.CrossRefGoogle Scholar
  9. 9.
    Kaprelyants A. S. and Kell D. B. (1992) Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow-cytometry. J. Appl. Bacteriol. 72, 410–422.Google Scholar
  10. 10.
    Gribbon L. T. and Barer M. R. (1995) Oxidativemetabolism in nonculturable Helicobacterpylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image-processing. Appl. Environ. Microbiol. 61, 3379–3384.PubMedGoogle Scholar
  11. 11.
    Kremer L., Baulard A., Estaquier J., Poulaingodefroy O., and Locht C., (1995) Green fluorescent protein as a new expression marker in mycobacteria. Mol. Microbiol. 17, 913–922.CrossRefPubMedGoogle Scholar
  12. 12.
    New R. R. C. (1990) Preparation of liposomes, in Liposomes: A Practical Approach (New R. R. C., ed.), IRL, Oxford, pp. 33–103.Google Scholar
  13. 13.
    Nwoguh C. E., Harwood C. R., and Barer M. R. (1995) Detection of induced beta-galactosidase activity in individual non-culturable cells of pathogenic bacteria by quantitative cytological assay. Mol. Microbiol. 17, 545–554.CrossRefPubMedGoogle Scholar
  14. 14.
    Barer M.R. (1997) Viable but non-culturable and dormant bacteria: time to resolve an oxymoron and a misnomer? J. Med. Microbiol. 46, 629–631.CrossRefPubMedGoogle Scholar
  15. 15.
    Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. R., and Barer M. R. (1998) Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek. 73, 169–187.CrossRefPubMedGoogle Scholar
  16. 16.
    Barer M. R. and Harwood C. R. (1999) Bacterial viability and culturability. Adv. Microb. Physiol. 41, in press.Google Scholar
  17. 17.
    Whiteley A. S., O’Donnell A. G., Macnaughton S. J., and Barer M. R. (1996) Cytochemical colocalization and quantitation of phenotypic and genotypic characteristics in individual bacterial cells. Appl. Environ. Microbiol. 62, 1873–1879.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Rory P. Cooney
  • Natalie J. Garton
  • Michael R. Barer

There are no affiliations available

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